Determinación del tropismo celular en la selección terapéutica. Aplicaciones clinicas de la

investigación de correceptores.

Eva Poveda

Servicio de Enfermedades Infecciosas

Hospital Carlos III, Madrid

Novel Antiretrovirals in Clinical Development

MatureMature

TNX-355CCR5 antagonistsCXCR4 antagonists

Maturation inhibitorsBevirimat

Raltegravir Elvitegravir

Entry inhibitors

Reverse transcriptase

inhibitors

virus

PIsIntegrase inhibitors

TNX-355CCR5 antagonistsCXCR4 antagonists

Maturation inhibitorsBevirimat

Raltegravir Elvitegravir

Entry inhibitors

Reverse transcriptase

inhibitors

virus

PIsIntegrase inhibitors

Viral Entry

Virus-CellFusion

gp41

gp120

V3 loop

CD4Binding

CD4

CellMembrane

CoreceptorBinding

CCR5/CXCR4(R5/X4)

HIV-1 Entry Inhibitors

Virus-CellFusion

gp41

gp120

V3 loop

CD4Binding

CD4

CellMembrane

CoreceptorBinding

CCR5/CXCR4(R5/X4)

CCR5 antagonistsMaraviroc (Selzentry)

VicrivirocPRO140

INCB9471

Enfuvirtide TRI-999 TRI-1144TNX-355

CXCR4 antagonists AMD070

Nt

gp120

CD4

+ inhibitor

- inhibitor

stem V3

ECL2

CCR5

V1/V2crown V3

Mecanismo de Acción de los antagonistas de CCR5

Chemokine coreceptors antagonists

HIV-1 co-receptor usage

CXCR4 CCR5CD4

Lineas celulares T Linfocitos primarios Monocitos/macrofagos

R5(NSI)

X4(SI)

R5(NSI)

R5-TropicX4-Tropic

DM

HIV Tropism and Disease Progression

R5 Mixed/Dual

Imm

un

e fu

nct

ion

Time

Am

ou

nt o

f virus

Limit of tropism assay detection

X4↑

Dual-tropic HIV

R5-tropic

X4-tropic

Cross-sectional Canadian study of 979 patients beginning triple therapy

• Strong association between presence of D/M or X4 virus and baseline CD4+ cell count

• Proportion of D/M and X4 virus ranging from < 10% at CD4+ cell count ≥ 200 cells/mm3 to > 50% at CD4+ cell count < 25 cells/mm3

• D/M or X4 virus progressively more likely in each lower CD4+ cell stratum

BL CD4+cell count, cells/mm3

R5 virus, %

D/M or X4 virus, %

> 500 93 7

350-499 91 9

200-349 91 9

100-199 72 28

50-99 74 26

25-49 69 31

< 25 46 54

Brumme ZL, et al. J Infect Dis. 2005;192:466-474.

Association between tropism and BL CD4+ confirmed

Tropism confirmed as a marker of HIV progresion

0.6

0.4

0.5

Kaplan-Meier curves showing progression to AIDS for patients with R5 or D/M virus

• BL tropism measured in 126 children and adolescents

• D/M virus at BL associated with lower BL CD4+ cell count and higher VL

• BL D/M virus associated with 3.8-fold higher risk of progression to AIDS

R5 virus

D/M virus

0 1 2 3 4 5 6 7 80.0

0.10.2

0.3

0.70.8

0.9

1.0

Pro

po

rtio

n A

IDS

fre

e

Time, years

Daar ES, et al. ICAAC 2003. Abstract 1722c.Daar ES, et al. Clin Infect Dis. 2007;45:643-649.

Conclusion: coreceptor tropism independently influences natural history of HIV disease.

1. Brumme ZL, et al. J Infect Dis. 2005;192:466-474. 2. Moyle GJ, et al. J Infect Dis. 2005;191:866-872. 3. Demarest J, et al. ICAAC 2004. Abstract H-1136. 4. Coakley E, et al. International Workshop on Targeting HIV Entry 2006. Abstract 8.

82%

81%

88%

85%

HOMER cohort[1]

(N = 979)

Chelsea and Westminster cohort[2]

(N = 402)

Demarest et al.[3]

(N = 299)

MERIT cohort[4]

(N = 1428)

18%

19%

12%

15%

< 1%

< 1%

< 1%

R5 D/M X4

HIV Tropism in Antiretroviral-Naive Populations

R5-only virus in 80% to 90% of patients, with D/M or X4 virus in remainder

50%

50%

59%

56%

TORO 1 and 2 ENF trials[1]

(N = 612)

ACTG A5211[2]

(N = 391)

SCOPE cohort[3]

(N = 186)

48%

46%

39.5%

41%

4%

0.5%

1. Melby J, et al. J Infect Dis.2006;194:238-246. 2. Wilkin TJ, et al. Clin Infect Dis. 2007;44:591-595. 3. Hunt PW, et al. J Infect Dis. 2006;194:926-930. 4. Coakley E, et al. International Workshop on Targeting HIV Entry 2006. Abstract 8.

MOTIVATE 1 and 2 MVC trials[4]

(N = 2560)

2%

3%

R5-only virus in 50% to 60% of patients, with D/M or X4 virus in remainder

R5 D/M X4

HIV Tropism in Antiretroviral-Experienced Populations

MVC fase 2b/III para estudiar la eficacia y la eficiencia de MVC en pacientes pre-tratados

infectados con variantes D/M

Mayer et al. XVI International AIDS Conference. Toronto, 2006 [THLB0215]

Chemokine coreceptors antagonists

+62+60+36CD4 change from baseline

30.8

26.9

24.6

21.1

24.1

15.5

HIV RNA <400 (%)

HIV RNA <50 (%)

-1.20-0.91-0.97

Mean decrease in HIV-1 RNA (log)

MVC BID +OBT

n=52

MVC QD + OBT

n=57

Placebo+OBT

n= 58

Treated patients with D/M-tropic HIV-1

Few data that relate to virtual and real phenotype

Idenfification of residues in V3 that strongly influence the viral co-receptor usage.

V3 phenotype prediction

Threshold for detection of X4 viruses in mixed population (R5+X4)

PhenoscriptTM (Eurofins-Viralliance, Kalamazoo, MI, USA) TrofileTM (Monogram Biosciences, San Francisco, CA, USA)

Recombinant viruses

Different levels of co-receptors between cell lines and natural targets of HIV

Ability of primary or recombinant virus isolates to replicate in cell lines that express CCR5 or CXCR4 receptors on their surface

Cell lines

To obtain viral stocksAbility of virus isolates to form syncitia in MT-2 cells

MT-2

LimitationsMethodologyAssays

Tools for viral tropism determination

Poveda et al. AIDS. 2006;20:1359-1367.

HIV-1 Coreceptor Tropism Assay

CD4+CCR5+

Infection

CD4+CXCR4+

++++

HIV genomicluc vector

HIV Envexpression

vector

Put into cell linewhere HIV can

replicate

Viral pseudotypes

Whitcomb J, et al. Antimicrob Agents Chemother. 2007;51:566-575.

0

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

99:1 95:5 90:10 50:50 10:90 5:95 1:99 controls

X4

R5

High sensitivity for detecting minoritary populations

1%

vira

l pro

duct

ion

(RLU

)

Tropism Report

Few data that relate to virtual and real phenotype

Idenfification of residues in V3 that strongly influence the viral co-receptor usage.

V3 phenotype prediction

Threshold for detection of X4 viruses in mixed population (R5+X4)

PhenoscriptTM (Eurofins-Viralliance, Kalamazoo, MI, USA) TrofileTM (Monogram Biosciences, San Francisco, CA, USA)

Recombinant viruses

Different levels of co-receptors between cell lines and natural targets of HIV

Ability of primary or recombinant virus isolates to replicate in cell lines that express CCR5 or CXCR4 receptors on their surface

Cell lines

To obtain viral stocksAbility of virus isolates to form syncitia in MT-2 cells

MT-2

LimitationsMethodologyAssays

Tools for viral tropism determination

Poveda et al. AIDS. 2006;20:1359-1367.

Predicción del tropismo en base a la secuencia genética de V3

Identificación de residuos de aa críticos relacionados con el tropismo

Websites de acceso público que predicen el uso del correceptor a partir de la secuencia de nucleótidos o aa de V3: http://genomiac2.ucsd.edu:8080/wetcat/v3.html http://www.geno2pheno.org http://ubik.microbiol.washington.edu/computing/pssm

Loop V3

11/25 Rule: basic aa (R or K) at 11 or 25 position of the V3 region ------ CXCR4 co-receptor usage.

Net Charge Rule: (K+R) – (D+E) 5 ------- CXCR4 co-receptor usage11

25

Low et al. AIDS. 2007;21.

Set of 920 clinical samples HOMER cohort (drug-naïve patients)

195978 Phenoscript

Env AssayTM

8.95.185.9236083PSSM

243.871.8394483Geno2pheno

26.92.570.5404383 Webcat (SVM)

DiscordanceX4/R5† (%)

Discordance R5/X4* (%)

Concordance(%)

X4R5No.Predictor

*Samples informed as R5 by genotypic methods and X4 by phenotype (Phenoscript Env AssayTM) †Samples informed as X4 by genotypic methods and R5 by phenotype.

Prediction of HIV-1 co-receptor use using genotypic and phenotypic methods

Poveda et al. AIDS. 2007;21:1487-1489.

195978 Phenoscript

Env AssayTM

88.178.985.9236083PSSM

67.888.871.8394483Geno2pheno

64.489.470.5404383 Webcat (SVM)

Specificity (%)Sensitivity (%)Concordance

(%)X4R5No.Predictor

*Samples informed as R5 by genotypic methods and X4 by phenotype (Phenoscript Env AssayTM) †Samples informed as X4 by genotypic methods and R5 by phenotype.

Prediction of HIV-1 co-receptor use using genotypic and phenotypic methods

Poveda et al. AIDS. 2007;21:1487-1489.

Combination of bioinformatic tools to infer HIV-1 co-receptor usage may be used as a screening strategy to determine tropism

Set of 200 samples from several database: 60% R5-tropic, 40 % X4-tropic

Sensitivity (%) Specificity (%)

SVM 98.8 62.5

GENO2PHENO 91.2 86.6

PART 83.8 81.7

PSSM 82.5 97.5

C4.5 82.5 97.5

CHARGE RULE 75 90.8

C4.5+8-12 70 97.5

Chueca et al. 2007 (in press).

73887761907471.388.676.5SVMgeno2pheno

92.58491.238.410062.583.888.685.2PSSMsinsi

88928953906982.591.485.2PSSMX4R5

92929230704782.585.783.2Charge Rule

92808930905682.582.982.6PART

941009523302682.58081.7C4.5 only with p8-12

88969023503477.582.979.1C4.5

858083461006978.885.780.9SVM

Non-BBTotalNon-BBTotalNon-BBTotal

SpecificitySensitivityConcordanceBioinformatics method

Garrido et al. J Clin Virol. 2007 (in press)

Evaluation of eight different bioinformatics tools to predict HIV-1 tropism in different subtypes

Set of 150 clinical samples: 115 non-B subtypes (54.3% ARV-experienced) 35 B subtypes (82.9% ARV-experienced)

Skrabal et al, J Clin Microbiol 2007; 45:279-84.

Degree of correlation between two phenotypic assays (Trofile vs. Phenoscript®ENV assays)

Set of 74 clinical samples

Degree of concordance 85.1% between both phenotypic assays 86.5% between Trofile/SVM 79.7% between Phenoscript®ENV assay/SVM

Chemokine coreceptors antagonists

Resistance to CCR5 antagonists

Outgrowth of X4 virus that pre-exits as a minority population below the level of assay detection.

Mutations in the HIV-1 gp120 molecule that allow the virus to bind to R5 receptors in the presence of drug.

Mutations in V3 loop of gp120 associated with MVC resistance but different pattern of amino acid changes between patients

Nelson et al, 14th CROI, 2007.

R5 ------> DM or X4 64% 5%

MVC Placebo

Clonal and phylogenic analyses of 20 pts (16 MVC, 4 placebo) suggest D/M virus predominantly from preexisting population

• Clinical implications remain to be fully defined

R5R5D/MD/MX4X4Nonfunctional cloneNonfunctional clone

0 100 200 300

TreatmentStart

R5 R5 D/M D/M D/M D/M D/M D/M R5 R5

Failure

TreatmentStop

Time since first administration (days)

Emergence of D/M Virus on CCR5 Antagonist Therapy

AgradecimientosHospital Carlos III:

Sección de Laboratorio:

Verónica Briz María del Mar González Carolina GarridoAngélica CorralNatalia ZahoneroCarmen de Mendoza

Sección Clínica:

Pablo Labarga Pilar García GascoFrancisco BlancoVicente SorianoJuan González-Lahoz

Eurofins-Viralliance, MI, USA

Katharina Skrabal

Vanessa Roulet

Jean-Louis Faudon

Hospital Univ. San Cecilio, Granada

Natalia Chueca

Federico García

Hosp. Xeral, Santiago de Compostela

Antonio Aguilera